Interphotoreceptor retinoid-binding protein (IRBP) has been in existence at least since the appearance of vertebrates. IRBP facilitates the uptake of all-trans-retinol into retinal ester stores in the retinal pigment epithelium. IRBP also facilitates the regeneration of opsin to rhodopsin in the retinal photoreceptor outer segment. The aims of this study are to define the parts of the protein that bind ligands. Structural studies revealed that the IRBP gene is unusual, having far fewer single polypeptide that contains a four-fold repeat structure. The first exon is extremely long and encodes all of the first three repeats and part of the fourth. Each repeat is about 300 amino acids long. Within the repeats there are probably three Domains and three interdomain Linkers. Certain biochemical analyses suggest that there is one retinoid binding site in the third repeat and perhaps another in the further repeat. Xenopus IRBP fourth repeat can bind all-tans-retinol (ATR), but analysis of binding data suggests that one repeat must interact with another copy of the same to form a dimeric molecule before it can bind a single molecule of ATR. In this proposal we seek funds to study the parts of this protein that bind retinoids and fatty acids. IRBP binds these ligands and protects them from degradation. That IRBP sequence changes cause no retinal degenerations, suggests that IRBP may have essential functions in other parts of the body. Another interpretation is that the multirepeat structure within IRBP prevents insufficient to cause complete loss of function of this four repeat protein. We plan to dissect IRBP into minimum functional units by the use of the previously defined structural domains and the baculovirus and bacterial expression systems developed for the expression of IRBP. These will be assayed biochemically and physiologically in vitro. We also will make several points mutations to the IRBP repeat and test the consequences of these changes. Key to this investigation is that we use the human cDNAs, and we will produce and test the human protein. This offers us the first chance to biochemically and physiologically study the medically relevant and useful material. We can directly compare human IRBP to the more frequently studied bovine IRBP.